Reduced folates are a family of vitamins that participate as cofactors in one-carbon transfer reactions that are involved in de novo synthesis of purines and thymidylate; synthesis of the amino acids serine, glycine, and methionine; degradation of histidine; scavenging of one-carbon metabolites such as formaldehyde and formate [1]. Folates themselves cannot be synthesized de novo by humans and thus must be obtained from dietary sources and by release from autotrophic enteric bacteria. Dietary and bacterially-derived folates are absorbed in the gut and transported as 5-methyltetrahydrofolate in the blood to the tissues. Once at the tissues, two general transport systems are available to cells for internalizing folates: (1) the folate-binding protein (FBP) family of endocytic, unidirectional, membrane receptor transporters; and (2) the reduced folate carrier (RFC; SLC19A1) carrier-mediated, bi-directional facilitated diffusion system [2].
Protein expression of these transporters is tissue-dependent. The RFC is expressed in most, if not all, tissues [3], while expression of the various FBP is limited to a few tissues [4]. Because of its wide distribution and its high capacity, the RFC is believed to be the primary means for transport of folates [2]. Of interest is that under most conditions, expression of the RFC is relatively constant in tissues in which it is expressed, although the levels expressed in different tissues vary widely. RFC activity may increase in acute folate deficiency [5], however; the mechanism of this increase is unknown. It has been suggested, based on the activation of human RFC promoter constructs by ectopic expression of specific transcription factors [6], that RFC expression could be transcriptionally regulated. However, data to support such regulation under physiological conditions is limited. Elucidation of mechanisms for regulating the transport of natural folates would be of fundamental interest.
Antifolates are antagonists of the action of the folate family of essential human vitamins, all of which are derived from the folic acid structure. The most commonly used antifolate in humans is currently methotrexate (MTX). However recently, two new antifolates with the same and/or different mechanisms of inhibiting folate metabolism have entered limited clinical use. These are raltitrexed (Tomudex; AstraZeneca) and pemetrexed (ALIMTA; Eli Lilly). MTX is used to treat a number of pathological conditions, including cancer, rheumatoid arthritis, psoriasis, and graft-versus-host-disease following bone marrow transplantation. The new antifolates are currently only approved to treat specific cancers (colon cancer and mesothelioma), but are undergoing clinical trial in tumors of other organ sites and in other diseases. A large number of antifolates have been made and tested preclinically; a number of these are now in clinical trial
Antifolates that closely resemble the folates structurally and which include the single glutamate (Glu) moiety that occurs in folates are termed “classical” antifolates. Classical antifolates including methotrexate (MTX), ZD1694, and pemetrexed are primarily transported into human cells by the equilibrative reduced folate carrier (RFC) and/or FBP [7]. Transport by tumors can be limiting to the therapeutic effect of antifolates. Once transported, classical antifolates are metabolized by folylpolyglutamate synthetase (FPGS) to poly(γ-glutamyl) forms, typically containing 1-7 additional glutamates in gamma-linkage. The polyglutamates are better retained within cells than are monoglutamates and provide a reservoir of drug that continues to act after extracellular drug declines or is removed. In addition, polyglutamyl antifolates may also be significantly more potent as inhibitors of their respective target enzymes.
Discovery of mechanisms by which antifolate transport could be increased in tumor cells might lead to greater therapeutic benefit from clinical use of current and future antifolates. In addition, metabolism of classical antifolates to their poly(γ-glutamate) forms by folylpolyglutamate synthetase is often limited by transport. Since polyglutamyl antifolates are better retained and are often more potent inhibitors of their target enzyme than is the parent monoglutamate, increased transport could also lead to enhanced synthesis of these important metabolites. This could be especially critical in childhood acute lymphoblastic leukemia where clinical correlations have shown that the median difference in MTX polyglutamate (MTXGn) accumulation between patients who respond to MTX-containing therapy and those who do not respond is only about three-fold. Increasing uptake of MTX even three-fold could increase MTXGn synthesis and might thus increase the number of long-term survivors.